Compositions and methods for modulating thrombin generation

ABSTRACT

Factor V peptides and methods of use thereof are disclosed.

This application is a divisional of U.S. patent application Ser. No.14/250,840, filed Apr. 11, 2014, which is a continuation-in-part ofPCT/US2012/060232, filed on Oct. 15, 2012, which claims priority under35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.61/546,752, filed Oct. 13, 2011. The foregoing applications areincorporated by reference herein.

This invention was made with government support under Grant NumbersR01HL088010 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and hematology.More specifically, the invention provides novel Factor V peptides andmethods of using the same to modulate the coagulation cascade inpatients in need thereof.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

In response to vascular injury such as a cut, coagulation enzymes areactivated in a stepwise manner, ultimately resulting in the formation ofa blood clot at the site of injury. Thrombin is generated from itsinactive precursor prothrombin in the final step of this cascade andsubsequently produces the fibrous clot. Prothrombinase, the enzymecomplex that activates thrombin, consists of the protease Factor Xa(FXa) and its non-enzymatic cofactor, activated Factor V (FVa), whichassemble on phospholipid membrane surfaces near the injury. FVa iscritical for thrombin generation, as FXa has very little activity in theabsence of FVa. Like other proteins of the coagulation cascade, theactive cofactor FVa is generated from an inactive precursor, Factor V(FV) which is an inactive procofactor. FV activation occurs by removalof a large inhibitory “B” domain as two fragments (˜71 kDa and ˜150 kDa)that maintains FV in an inactive state. Accordingly, the inhibition ofFV activation or stabilization of the inactive procofactor state isdesired in order to reduce improper or unwanted thrombin generation andclot formation.

SUMMARY OF THE INVENTION

In accordance with the instant invention, Factor V peptides for themodulation of thrombin generation are provided. In a particularembodiment, the peptide has at least 80% homology with SEQ ID NO: 1, 2,3 or 4. Nucleic acid molecules encoding the peptides are alsoencompassed by the instant invention. Compositions comprising at leastone peptide and/or nucleic acid of the instant invention and at leastone pharmaceutically acceptable carrier are also provided. Thecompositions may further comprise at least one other anti-thrombosiscompound.

According to another aspect of the instant invention, methods forinhibiting, treating, and/or preventing clot formation in a subject inneed thereof are provided. Methods for inhibiting, treating, and/orinhibiting a hemostasis disorder in a patient in need thereof areprovided. In a particular embodiment, the methods comprise administeringto the subject at least one composition of the instant invention. Themethods may further comprise administering at least one additionalanti-thrombosis compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of Factor V with the amino acid sequence ofa conserved basic region within the B domain (SEQ ID NO: 5). A sequencealignment of the conserved basic region across various species is alsoprovided. Sequences are SEQ ID NOs: 6-15, from top to bottom.

FIGS. 2A-2D provide graphs showing that a FV basic region peptideinhibits multiple cofactor-like FV variants. FIG. 2A is a graph of atitration of the FV basic peptide (FVBR) inhibiting activation ofprothrombin by FXa/FVDT but not FXa/FVa. FIG. 2B shows the activity ofFVDT and other variants in clotting assays in the presence of FVBR orthe control peptide (s46). FIG. 2C provides schematics of various FV-810variants and their cleavage products. FIG. 2C also provides a graph ofthe activity of FVDT and these variants in clotting assays in thepresence or absence of FVBR and the presence or absence of thrombin(IIa). FIG. 2D shows the clot time of normal human plasma withincreasing amounts of FVBR.

FIGS. 3A-3C provide graphs demonstrating that a FV basic region peptidebinds FVDT and disrupts binding of FXa to FVDT but not to FVa. FIG. 3Ashows the binding of fluorescently-labeled FXa to FVDT on liposomes inthe presence or absence of FVBR. FIG. 3B shows the binding of FXa toFVDT or FVa in the presence of different concentrations of FVBR. FIG. 3Cshows the direct binding of fluorescently labeled FVBR to FVDT.

FIGS. 4A-4B show that FVBR delays the proteolytic activation of FV bythrombin. FIGS. 4A and 4B provide images of the cleavage of FV over timein the absence or presence of FVBR, respectively. B+LC=B domain andlight chain. HC=heavy chain. LC=light chain.

FIG. 5 provides a schematic of Factor V with the amino acid sequences ofa conserved basic region (SEQ ID NO: 6) and a conserved acidic region(SEQ ID NO: 16) within the B domain. A sequence alignment of theconserved acidic region across various species is also provided.Sequences are SEQ ID NOs: 16-25, from top to bottom.

FIG. 6 provides a graph of the activity of various FV variants.

FIG. 7A shows a sequence alignment of the tissue factor pathwayinhibitor (TFPI) C-terminal tail (SEQ ID NO: 4) and FVBR (SEQ ID NO: 2).FIG. 7B provides a graph of the inhibition of FVDT by the FVBR or TFPIpeptide. FIG. 7C shows the direct binding of TFPI to FVDT. FIG. 7D showsa competition assay of unlabeled FVBR or TFPI with Oregon Green® 488FVBR for binding with FVDT.

FIGS. 8A and 8B show the specific clotting activity measured inFV-deficient plasma supplemented with 0.25 nM FV-810 (FIG. 8A) or rFVa(FIG. 8B) and the indicated peptides at 5 μM. Ac-BR=acetylated BRpeptide.

FIG. 9 shows the direct binding of the BR peptide to FV-810. FV-810 wastitrated into reaction mixtures containing 20 nM (●) or 40 nM (∘)OG488-BR peptide and 50 μM PCPS in assay buffer at 25° C. Lines weredrawn after analysis to independent, non-interacting sites with thefitted constants Kd=2.07±0.2 nM and n=1.27±0.02 mol of FV-810/mol ofOG488-BR at saturation. Control experiments were performed by titratingFV-810 into buffer containing 10 mM EDTA (X) or by titrating rFVa (▴).Inset, the unlabeled BR peptide was titrated into reaction mixturescontaining 30 nM OG488-BR, 20 nM FV-810, and 50 μM PCPS. The fittedconstants for the unlabeled BR peptide were determined as Kd=2.1±0.2 nMand n=1.0±0.06 mol of BR/mol of FV-810 assuming the constants determinedabove for OG488-BR.

FIGS. 10A and 10B show sedimentation velocity of the BR peptide. Thesedimentation velocity of 5 μM QSY7-BR was measured either alone (FIG.10A) or in the presence of 7 μM FV-810 (FIG. 10B). The panels show 14scans taken at 8-minute intervals.

FIG. 11A shows human (●), bovine (♦), lizard (▴), and zebrafish (▪) BRpeptides titrated into reactions containing 1.4 μM prothrombin, 3 μMDAPA, 50 μM PCPS, and 0.1 nM FV-810 in assay buffer at 25° C.Prothrombin activation was measured. FIG. 11B shows human (●) and bovine(♦) BR peptides titrated into reactions containing 30 nM OG488-BR, 20 nMFV-810, and 50 μM PCPS at 25° C. Fluorescence anisotropy was measured,and equilibrium binding constants were determined assuming astoichiometry of 1 mol of FV-810/mol of BR peptide: the human BR,Kd=2.2±0.2 nM; and the bovine BR, Kd=28.3±0.6 nM.

FIGS. 12 A and 12B show the competitive binding of the BR peptide andFXa to FV-810. FIG. 12A: FXaS195A (●) or zymogen FXS195A (▴) wastitrated into reaction mixtures containing 30 nM OG488-BR, 20 nM FV-810,and 50 μM PCPS in assay buffer at 25° C. Changes in OG488-BR anisotropywere measured, and lines were drawn with the fitted constants Kd=1.8±0.2nM for FXa and n=1.1±0.07 mol of FXa/mol of FV-810. FIG. 12B: Reactionscontaining 1.4 μM prethrombin-2, 3 μM DAPA, 50 μM PCPS, 5 nM FV-810, and0 nM (▪), 125 nM (●), 250 nM (▴), 500 nM (♦), or 1 μM (▾) BR peptidewere prepared in assay buffer at 25° C. Reactions were initiated by theaddition of 1-50 nM FXa, and thrombin generation was monitored.Experimental data were fitted to a model for tight binding withcalculated values of Kd=2.0±0.2 nM for FXa and Kd=34.2±3.6 nM for theBR.

FIG. 13 provides an image of a gel of reactions containing 600 nMFV-810, FV-810^(R709Q), FV-810^(R1545Q), FV-810^(QQ), or rFVa incubatedfor 15 min at 37° C. with buffer or 10 nM thrombin and then quenchedwith 20 nM hirudin. Samples were resolved by 4-12% gradient SDS-PAGEunder reducing conditions and stained with Coomassie Brilliant Blue.

FIGS. 14A and 14B show the direct binding of OG488-BR tothrombin-cleaved FV-810 variants. FV-810 (●), FV-810^(R709Q) (▴),FV-810^(R1545Q) (♦), and FV-810^(QQ) (▪) were pretreated with buffer(FIG. 14A) or thrombin (FIG. 14B). Quenched FV-810 species were titratedinto reaction mixtures containing 30 nM OG488-BR and 50 μM PCPS in assaybuffer, and changes in the fluorescence anisotropy signal were measured.Binding constants were calculated assuming a stoichiometry of n=1 of molFV-810/mol of OG488-BR: FV-810, Kd=2.1±0.3 nM; FV-810^(R709Q),Kd=7.1±1.6 nM; FV-810^(R1545Q), Kd=2.0±0.4 nM; and FV-810^(QQ),Kd=0.31±0.29 nM. After incubation with thrombin, neither FV-810 norFV-810^(R709Q) had detectable binding to OG488-BR, whereas FV-810^(QQ)bound with a calculated Kd of 1.3±0.1 nM, and FV-810^(R1545Q) bound witha Kd of 30.3±4.1 nM.

DETAILED DESCRIPTION OF THE INVENTION

Herein, peptides that inhibit the generation of the clotting enzymethrombin are provided. Specifically, a region of approximately 50 aminoacids within the FV B domain (963-1008) has been identified that isrequired to keep FV inactive. This region is enriched with the basicamino acids arginine and lysine, giving it a strong positive net chargeat physiological pH. Removal of this region (the FV basic region)switches FV from an inactive procofactor to an active, FVa-like cofactorthat rescues thrombin generation. Without being bound by theory, thedata provided herein indicate that the basic region functions at leastby interacting with a highly acidic region at the C-terminal end of theB domain (1493-1537) and that this interaction maintains FV in aninactive state. When added to reactions as a separate peptide, the FVbasic region inhibits cofactor-like FV variants that contain the acidicregion, rendering them procofactor-like and inhibiting thrombingeneration. Furthermore, the FV basic region peptide substantiallyimpairs the ability of thrombin to proteolytically convert FV to FVa, animportant feedback step of the coagulation cascade in vivo. These dataindicate that the FV basic region peptides of the instant inventioneffectively inhibit FV by maintaining the inactive, procofactor-likestate, thereby reducing thrombin generation and clot formation.

Peptides

Peptides of the present invention may be prepared in a variety of ways,according to known methods. The proteins may be purified fromappropriate sources, e.g., transformed bacterial or animal culturedcells or tissues. The availability of nucleic acid molecules encodingthe peptides of the instant invention enables production of the proteinusing in vitro expression methods and cell-free expression systems knownin the art. In vitro transcription and translation systems arecommercially available, e.g., from Promega Biotech (Madison, Wis.) orGibco-BRL (Gaithersburg, Md.).

Larger quantities of peptides of the instant invention may be producedby expression in a suitable prokaryotic or eukaryotic system. Forexample, part or all of a DNA molecule encoding for the peptides of theinstant invention may be inserted into a plasmid vector adapted forexpression in a bacterial cell, such as E. coli. Such vectors comprisethe regulatory elements necessary for expression of the DNA in the hostcell positioned in such a manner as to permit expression of the DNA inthe host cell. Such regulatory elements required for expression includepromoter sequences, transcription initiation sequences and, optionally,enhancer sequences.

Peptides of the instant invention produced by gene expression in arecombinant prokaryotic or eukaryotic system may be purified accordingto methods known in the art.

A commercially available expression/secretion system can be used,whereby the recombinant protein is expressed and thereafter secretedfrom the host cell, and readily purified from the surrounding medium.The recombinant protein may also be purified by affinity separation,such as by immunological interaction with antibodies that bindspecifically to the recombinant protein or nickel columns for isolationof recombinant proteins tagged with 6-8 histidine residues at theirN-terminus or C-terminus. Alternative tags may comprise the FLAG epitopeor the hemagglutinin epitope. Such methods are commonly used by skilledpractitioners.

Peptides of the instant invention may also be made by peptide synthesis.For example, the peptides may be made by liquid-phase peptide synthesisor solid-phase peptide synthesis (SPPS).

Peptides of the instant invention, prepared by the aforementionedmethods, may be analyzed according to standard procedures. For example,such protein may be subjected to amino acid sequence analysis, accordingto known methods.

Examples of amino acid sequences of the peptides of the instantinvention include SRAWGESTPLANKPGKQSGHPKFPRVRHKSLQVRQDGGKSRLKKSQFLIKTRKKKKEK (SEQ ID NO: 1), KPGKQSGHPKFPRVRHKSLQVRQDGGKSRLKKSQ FLIKTRKKKKEK(SEQ ID NO: 2), RQDGGKSRLKKSQFLIKTRKKKKEK (SEQ ID NO: 3), andKKGFIQRISKGGLIKTKRKRKKQRVK (SEQ ID NO: 4). The amino acid sequence ofthe peptides of the instant invention may have at least 75%, 80%, 85%,90%, 95%, 97%, 99%, or 100% homology (identity) with SEQ ID NO: 1, 2, 3or 4, particularly at least 90% homology. In a particular embodiment,the peptide is a fragment of SEQ ID NO: 1, 2, 3, or 4. For example, thepeptide fragment may comprise at least about 20, about 25, about 30,about 35, about 40, about 45, about 50, or about 55 contiguous aminoacids of SEQ ID NO: 1, 2, 3, or 4. In a particular embodiment, thepeptide fragment is a fragment of SEQ ID NO: 1, but comprises SEQ ID NO:2 or 3.

In a particular embodiment, the peptide of the instant invention has alength of about 10 to about 100, about 20 to about 80 amino acids, about20 to about 60, about 25 to about 60, about 30 to about 70 amino acids,or about 40 to about 60 amino acids. As stated hereinabove, the peptidesof the instant invention may comprise a sequence having at least 75%,80%, 85%, 90%, 95%, 97%, 99%, or 100% homology (identity) with SEQ IDNO: 1, 2, 3, or 4 or a fragment thereof. The peptides of the instantinvention may extend beyond SEQ ID NO: 1, 2, 3, or 4 or a fragmentthereof at either the amino or carboxy terminus. The sequence extensionat either end may be of any sequence. In a particular embodiment, theextension sequence comprises basic amino acids. In a particularembodiment, the extension sequence corresponds to the B domain sequenceof Factor V (e.g., for SEQ ID NO: 3) or TFPI (e.g., for SEQ ID NO: 4).For example, if the sequence extends three amino acids C-terminal to SEQID NO: 1, the sequence of the three amino acid extension may be HTH,which corresponds to amino acids 1009-1011 of the B domain.

The peptides of the instant invention may contain at least onesubstitution, addition, or insertion to the amino acids of SEQ ID NO: 1,2, 3, or 4. These substitutions may be conservative—i.e., similar to theamino acid present in SEQ ID NO: 1, 2, 3 or 4 (e.g., an acidic aminoacid in place of another acidic amino acid, a basic amino acid in placeof a basic amino acid, a large hydrophobic amino acid in place of alarge hydrophobic, etc.). The substitutions may comprise amino acidanalogs and mimetics. In a particular embodiment, the substitution(s)and/or addition(s) increases the number of basic amino acids present inthe peptide. In a particular embodiment, the substitutions increase theaffinity of the peptide for the conserved acidic region of the B domain(see FIG. 5).

The peptides of the instant invention may have capping, protectingand/or stabilizing moieties, for example at the C-terminus and/orN-terminus. Such moieties are well known in the art and include, withoutlimitation, amidation or esterification of the carboxy-terminal end andacylation or acetylation of the amino-terminal end. The peptide may alsobe PEGylated. The peptide may also be lipidated or glycosylated at anyamino acid (i.e., a glycopeptide). The peptides of the instant inventionmay also comprise at least one D-amino acid instead of the nativeL-amino acid. The peptides may comprise only D-amino acids.

The peptides of the instant invention can be based on a Factor V fromany species, particularly a mammalian Factor V, more particularly ahuman Factor V. FIG. 1 provides examples of the Factor V basic domainfrom various species. GenBank Accession No. NP_000121.2 provides anexample of the wild-type human FV precursor protein wherein amino acids1-28 are a signal peptide that is cleaved and numbering begins atresidue 29. The peptides of the instant invention can also be based on aTFPI from any species, particularly a mammalian TFPI, more particularlya human TFPI. GenBank Accession No. NP_006278.1 provides an example ofthe wild-type human TFPI precursor protein wherein amino acids 1-28 area signal peptide that is cleaved and numbering begins at residue 29.

Compositions comprising at least one peptide and at least one carrierare also encompassed by the instant invention. Except insofar as anyconventional carrier is incompatible with the peptide to beadministered, its use in the pharmaceutical composition is contemplated.In a particular embodiment, the carrier is a pharmaceutically acceptablecarrier for intravenous administration.

Nucleic Acid Molecules

Nucleic acid molecules encoding the peptides of the invention may beprepared by any method known in the art such as (1) synthesis fromappropriate nucleotide triphosphates or (2) isolation and/oramplification from biological sources. The availability of nucleotidesequence information enables preparation of an isolated nucleic acidmolecule of the invention by oligonucleotide synthesis. Indeed,knowledge of the amino sequence is sufficient to determine an encodingnucleic acid molecule. Synthetic oligonucleotides may be prepared by thephosphoramidite method employed in the Applied Biosystems 38A DNASynthesizer or similar devices. The resultant construct may be purifiedaccording to methods known in the art, such as gel electrophoresis orhigh performance liquid chromatography (HPLC).

Nucleic acids of the present invention may be maintained in anyconvenient vector or viral vector, particularly an expression vector.Different promoters may be utilized to drive expression of the nucleicacid sequences based on the cell in which it is to be expressed.Antibiotic resistance markers are also included in these vectors toenable selection of transformed cells. Peptide encoding nucleic acidmolecules of the invention include cDNA, genomic DNA, DNA, RNA, andfragments thereof which may be single- or double-stranded. Thus, thisinvention provides oligonucleotides having sequences capable ofhybridizing with at least one sequence of a nucleic acid molecule of thepresent invention.

Also encompassed in the scope of the present invention areoligonucleotide probes which specifically hybridize with the peptideencoding nucleic acid molecules of the invention. Primers capable ofspecifically amplifying peptide encoding nucleic acids described hereinare also contemplated herein. Such oligonucleotides are useful as probesand primers for detecting, isolating or amplifying peptide encodingnucleic acids.

It will be appreciated by persons skilled in the art that variants(e.g., allelic variants) of Factor V sequences exist, for example, inthe human population, and may be taken into account when designingand/or utilizing oligonucleotides or peptides of the invention.Accordingly, it is within the scope of the present invention toencompass such variants, with respect to the peptide sequences disclosedherein or the oligonucleotides targeted to specific locations on therespective genes or RNA transcripts. Accordingly, the term “naturalallelic variants” is used herein to refer to various specific nucleotidesequences of the invention and variants thereof that would occur in ahuman population. The usage of different wobble codons and geneticpolymorphisms which give rise to conservative or neutral amino acidsubstitutions in the encoded protein are examples of such variants. Suchvariants would not demonstrate substantially altered activity or proteinlevels.

Compositions comprising at least one nucleic acid molecule of theinstant invention (e.g., a vector) and at least one carrier are alsoencompassed by the instant invention. The compositions of the instantinvention may be used, for example, as therapeutic and/or prophylacticagents (protein or nucleic acid) which modulate the blood coagulationcascade. It is demonstrated herein that the peptides can inhibit clotformation and effect hemostasis.

Uses

The instant invention encompasses methods of inhibiting and/orpreventing clot formation. The instant invention also encompassesmethods of treating and/or inhibiting hemostasis disorders, particularlydisorders with aberrant, excessive, or improper coagulation. Examples ofsuch hemostasis disorders include, without limitation, thrombosis, deepvenous thrombosis, thrombosis associated with cardiovascular disease,thrombosis associated with a malignancy, thrombosis resulting frominvasive surgical devices (e.g., catheters, cardiac catheter,intravascular catheter, intra-aortic balloon pump, coronary stent, orcardiac valve), thrombosis associated with autoimmune diseases (e.g.,lupus), thrombocytopenia (e.g., heparin-induced), stroke (e.g., embolicstroke, thrombotic stroke), coagulopathy. The peptides can providenecessary anticoagulant treatment for patients with disseminatedintravascular coagulation or consumptive coagulopathies arising from avariety of disease or disorder states. The peptides can providenecessary anticoagulant treatment for patients suffering from myocardialinfarction.

In a particular embodiment of the present invention, peptides of theinstant invention may be administered to a patient in a pharmaceuticallyacceptable carrier, particularly via intravenous injection. The peptidesof the instant invention may optionally be encapsulated into liposomesor mixed with other phospholipids or micelles to increase stability ofthe molecule. Peptides may be administered alone or in combination withother agents known to modulate hemostasis (e.g., agents which inhibitclot formation). For example, the compositions of the instant inventionmay be co-administered with other anti-thrombosis compounds. Examples ofanti-thrombosis compounds include, without limitation, vitamin Kantagonists (e.g., warfarin, acenocoumarol, dicumarol, phenprocoumon,related 4-hydroxycoumarin-containing molecules, phenindione, andinhibitors of vitamin K epoxide reductase), direct thrombin inhibitors(DTIs; e.g., hirudin, bivalirudin, lepirudin, argatroban, ximelagatran,melagatran, and dabigatran), Factor Xa inhibitors (e.g., heparin, lowmolecular weight heparin, certoparin, dalteparin, enoxaparin,nadroparin, tinzaparin, reviparin, parnaparin, bemiparin, fondaparinux,idraparinux, heparinoid, danaparoid, sulodexide, xabans, apixaban,betrixaban, edoxaban, otamixaban, and rivaroxaban), defibrotide, andanti-platelet agents (e.g., glycoprotein IIb/IIIa inhibitors (e.g.,abciximab, eptifibatide, tirofiban), ADP receptor/P2Y₁₂ inhibitors(e.g., thienopyridines (e.g., clopidogrel, prasugrel, and ticlopidine)and nucleotide/nucleoside analogs (e.g., cangrelor, elinogrel, andticagrelor); prostaglandin analogue (e.g., beraprost, prostacyclin,iloprost, and treprostinil); COX inhibitors (e.g., acetylsalicylicacid/aspirin, carbasalate calcium, indobufen, and triflusal);thromboxane inhibitors (e.g., thromboxane synthase inhibitors such asdipyridamole or picotamide) and receptor antagonists such asterutroban); phosphodiesterase inhibitors (e.g., cilostazol,dipyridamole, and triflusal). The anti-thrombosis compound may be in thesame composition comprising the peptide of the instant invention or maybe in a separate composition. The compositions may be administeredconcurrently or consecutively. Kits comprising at least one firstcomposition comprising at least one peptide of the instant invention andat least one second composition comprising at least one otheranti-thrombosis compound are encompassed by the instant invention.

An appropriate composition in which to deliver peptides may bedetermined by a medical practitioner upon consideration of a variety ofphysiological variables, including, but not limited to, the patient'scondition and hemodynamic state. A variety of compositions well suitedfor different applications and routes of administration are well knownin the art and are described hereinbelow.

The preparation containing the purified peptides contains aphysiologically acceptable matrix and is preferably formulated as apharmaceutical preparation. The preparation can be formulated usingsubstantially known prior art methods, it can be mixed with a buffercontaining salts, such as NaCl, CaCl₂, and amino acids, such as glycineand/or lysine, and in a pH range from 6 to 8. Until needed, the purifiedpreparation containing the peptide can be stored in the form of afinished solution or in lyophilized or deep-frozen form. In a particularembodiment, the preparation is stored in lyophilized form and isdissolved into a visually clear solution using an appropriatereconstitution solution. Alternatively, the preparation according to thepresent invention can also be made available as a liquid preparation oras a liquid that is deep-frozen. The preparation according to thepresent invention is especially stable, i.e., it can be allowed to standin dissolved form for a prolonged time prior to application.

Prior to processing the purified protein into a pharmaceuticalpreparation, the purified peptide may be subjected to the conventionalquality controls and fashioned into a therapeutic form of presentation.In particular, during the recombinant manufacture, the purifiedpreparation may be tested for the absence of cellular nucleic acids aswell as nucleic acids that are derived from the expression vector.

Peptide-encoding nucleic acids may be used for a variety of purposes inaccordance with the present invention. In a particular embodiment of theinvention, a nucleic acid delivery vehicle (i.e., an expression vector)for modulating blood coagulation is provided wherein the expressionvector comprises a nucleic acid sequence coding for a peptide of theinstant invention. Administration of peptide-encoding expression vectorsto a patient results in the expression of peptide which serves to alterthe coagulation cascade.

Expression vectors comprising peptide nucleic acid sequences may beadministered alone, or in combination with other molecules useful formodulating hemostasis. According to the present invention, theexpression vectors or combination of therapeutic agents may beadministered to the patient alone or in a pharmaceutically acceptable orbiologically compatible composition.

In a particular embodiment of the invention, the expression vectorcomprising nucleic acid sequences encoding the peptide is a viralvector. Viral vectors which may be used in the present inventioninclude, but are not limited to, adenoviral vectors (with or withouttissue specific promoters/enhancers), adeno-associated virus (AAV)vectors of multiple serotypes (e.g., AAV-2, AAV-5, AAV-7, and AAV-8) andhybrid AAV vectors, lentivirus vectors and pseudo-typed lentivirusvectors (e.g., Ebola virus, vesicular stomatitis virus (VSV), and felineimmunodeficiency virus (FIV)), herpes simplex virus vectors, vacciniavirus vectors, and retroviral vectors.

In a particular embodiment of the present invention, methods areprovided for the administration of a viral vector comprising nucleicacid sequences encoding peptides of the instant invention. Adenoviralvectors of utility in the methods of the present invention preferablyinclude at least the essential parts of adenoviral vector DNA. Asdescribed herein, expression of a peptide following administration ofsuch an adenoviral vector serves to modulate hemostasis. Recombinantadenoviral vectors have found broad utility for a variety of genetherapy applications. Their utility for such applications is due largelyto the high efficiency of in vivo gene transfer.

The expression vectors of the present invention may be incorporated intopharmaceutical compositions that may be delivered to a subject, so as toallow production of a biologically active peptide. In a particularembodiment of the present invention, pharmaceutical compositionscomprising sufficient genetic material to enable a recipient to producea therapeutically effective amount of a peptide can influence hemostasisin the subject. Alternatively, as discussed above, an effective amountof the peptide may be directly injected into a patient in need thereof.The compositions may be administered alone or in combination with atleast one other agent, such as a stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents which influence hemostasis.

In particular embodiments, the pharmaceutical compositions also containa pharmaceutically acceptable excipient/carrier. Such excipients includeany pharmaceutical agent that does not itself induce an immune responseharmful to the individual receiving the composition, and which may beadministered without undue toxicity. Pharmaceutically acceptableexcipients include, but are not limited to, liquids such as water,saline, glycerol, sugars and ethanol. Pharmaceutically acceptable saltscan also be included therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A thorough discussion ofpharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., Easton, Pa.).

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended therapeutic purpose.Determining a therapeutically effective dose is well within thecapability of a skilled medical practitioner using the techniques andguidance provided in the present invention. Therapeutic doses willdepend on, among other factors, the age and general condition of thesubject, the severity of the aberrant blood coagulation phenotype, andthe strength of the control sequences regulating the expression levelsof the peptide. Thus, a therapeutically effective amount in humans willfall in a relatively broad range that may be determined by a medicalpractitioner based on the response of an individual patient to peptidetreatment.

Direct delivery of the pharmaceutical compositions in vivo may generallybe accomplished via injection using a conventional syringe. Thecompositions of the instant invention may be delivered subcutaneously,epidermally, intradermally, intrathecally, intraorbitally,intramucosally, intraperitoneally, intravenously, intraarterially,orally, intrahepatically or intramuscularly. A clinician specializing inthe treatment of patients with blood coagulation disorders may determinethe optimal route for administration of the compositions of the instantinvention based on a number of criteria, including, but not limited to:the condition of the patient and the purpose of the treatment (e.g.,reduced blood coagulation).

Definitions

Various terms relating to the biological molecules of the presentinvention are used hereinabove and also throughout the specification andclaims.

With reference to nucleic acids of the invention, the term “isolatednucleic acid” is sometimes used. This term, when applied to DNA, refersto a DNA molecule that is separated from sequences with which it isimmediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it originates. For example,the “isolated nucleic acid” may comprise a DNA or cDNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into theDNA of a prokaryote or eukaryote.

With respect to RNA molecules of the invention, the term “isolatednucleic acid” primarily refers to an RNA molecule encoded by an isolatedDNA molecule as defined above. Alternatively, the term may refer to anRNA molecule that has been sufficiently separated from RNA moleculeswith which it would be associated in its natural state (i.e., in cellsor tissues), such that it exists in a “substantially pure” form.

The term “oligonucleotide” as used herein refers to sequences, primersand probes of the present invention, and is defined as a nucleic acidmolecule comprised of two or more ribo- or deoxyribonucleotides,preferably more than three. The exact size of the oligonucleotide willdepend on various factors and on the particular application and use ofthe oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such asappropriate temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product. The primer mayvary in length depending on the particular conditions and requirement ofthe application. For example, in diagnostic applications, theoligonucleotide primer is typically 10-30 or 15-25, or more nucleotidesin length. The primer must be of sufficient complementarity to thedesired template to prime the synthesis of the desired extensionproduct, that is, to be able to anneal with the desired template strandin a manner sufficient to provide the 3′ hydroxyl moiety of the primerin appropriate juxtaposition for use in the initiation of synthesis by apolymerase or similar enzyme. It is not required that the primersequence represent an exact complement of the desired template. Forexample, a non-complementary nucleotide sequence may be attached to the5′ end of an otherwise complementary primer. Alternatively,non-complementary bases may be interspersed within the oligonucleotideprimer sequence, provided that the primer sequence has sufficientcomplementarity with the sequence of the desired template strand tofunctionally provide a template-primer complex for the synthesis of theextension product.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 10-30 or 15-25,or more nucleotides, although it may contain fewer nucleotides. Theprobes herein are selected to be complementary to different strands of aparticular target nucleic acid sequence. This means that the probes mustbe sufficiently complementary so as to be able to “specificallyhybridize” or anneal with their respective target strands under a set ofpre-determined conditions. Therefore, the probe sequence need notreflect the exact complementary sequence of the target. For example, anon-complementary nucleotide fragment may be attached to the 5′ or 3′end of the probe, with the remainder of the probe sequence beingcomplementary to the target strand. Alternatively, non-complementarybases or longer sequences can be interspersed into the probe, providedthat the probe sequence has sufficient complementarity with the sequenceof the target nucleic acid to anneal therewith specifically.

With respect to single stranded nucleic acids, particularlyoligonucleotides, the term “specifically hybridizing” refers to theassociation between two single-stranded nucleotide molecules ofsufficiently complementary sequence to permit such hybridization underpre-determined conditions generally used in the art (sometimes termed“substantially complementary”). In particular, the term refers tohybridization of an oligonucleotide with a substantially complementarysequence contained within a single-stranded DNA molecule of theinvention, to the substantial exclusion of hybridization of theoligonucleotide with single-stranded nucleic acids of non-complementarysequence. Appropriate conditions enabling specific hybridization ofsingle stranded nucleic acid molecules of varying complementarity arewell known in the art.

For instance, one common formula for calculating the stringencyconditions required to achieve hybridization between nucleic acidmolecules of a specified sequence homology is set forth below (Sambrooket al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press):Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/#bp induplex

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C.with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C.

The stringency of the hybridization and wash depend primarily on thesalt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the probe with its target, thehybridization is usually carried out at salt and temperature conditionsthat are 20 25° C. below the calculated Tm of the hybrid. Washconditions should be as stringent as possible for the degree of identityof the probe for the target. In general, wash conditions are selected tobe approximately 12 20° C. below the Tm of the hybrid. In regards to thenucleic acids of the current invention, a moderate stringencyhybridization is defined as hybridization in 6×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C.,and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A highstringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. Avery high stringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.

The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated nucleic acid molecule of the invention.Alternatively, this term may refer to a protein that has beensufficiently separated from other proteins with which it would naturallybe associated, so as to exist in “substantially pure” form. “Isolated”is not meant to exclude artificial or synthetic mixtures with othercompounds or materials, or the presence of impurities that do notinterfere with the fundamental activity, and that may be present, forexample, due to incomplete purification, or the addition of stabilizers.

The term “vector” refers to a carrier nucleic acid molecule (e.g., DNA)into which a nucleic acid sequence can be inserted for introduction intoa host cell where it will be replicated. An “expression vector” is aspecialized vector that contains a gene or nucleic acid sequence withthe necessary regulatory regions needed for expression in a host cell.

The term “operably linked” means that the regulatory sequences necessaryfor expression of a coding sequence are placed in the DNA molecule inthe appropriate positions relative to the coding sequence so as toeffect expression of the coding sequence. This same definition issometimes applied to the arrangement of coding sequences andtranscription control elements (e.g. promoters, enhancers, andtermination elements) in an expression vector. This definition is alsosometimes applied to the arrangement of nucleic acid sequences of afirst and a second nucleic acid molecule wherein a hybrid nucleic acidmolecule is generated.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight the compound of interest (e.g., nucleic acid,oligonucleotide, protein, etc.), particularly at least 75% by weight, orat least 90-99% or more by weight of the compound of interest. Puritymay be measured by methods appropriate for the compound of interest(e.g. chromatographic methods, agarose or polyacrylamide gelelectrophoresis, HPLC analysis, and the like).

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, preservative(e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid,sodium metabisulfite), solubilizer (e.g., Tween 80, Polysorbate 80),emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), antimicrobial,bulking substance (e.g., lactose, mannitol), excipient, auxiliary agentor vehicle with which an active agent of the present invention isadministered. Pharmaceutically acceptable carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin. Water or aqueous saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin (Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington:The Science and Practice of Pharmacy, (Lippincott, Williams andWilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, MarcelDecker, New York, N.Y.; and Kibbe, et al., Eds., Handbook ofPharmaceutical Excipients, American Pharmaceutical Association,Washington.

As used herein, a “conservative” amino acid substitution/mutation refersto substituting a particular amino acid with an amino acid having a sidechain of similar nature (i.e., replacing one amino acid with anotheramino acid belonging to the same group). A “non-conservative” amino acidsubstitution/mutation refers to replacing a particular amino acid withanother amino acid having a side chain of different nature (i.e.,replacing one amino acid with another amino acid belonging to adifferent group). Groups of amino acids having a side chain of similarnature are known in the art and include, without limitation, basic aminoacids (e.g., lysine, arginine, histidine); acidic amino acids (e.g.,aspartic acid, glutamic acid); neutral amino acids (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan); amino acids having a polar side chain (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine); aminoacids having a non-polar side chain (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); amino acidshaving an aromatic side chain (e.g., phenylalanine, tryptophan,histidine); amino acids having a side chain containing a hydroxyl group(e.g., serine, threonine, tyrosine), and the like.

The following examples are provided to illustrate various embodiments ofthe present invention. The examples are illustrative and are notintended to limit the invention in any way.

EXAMPLE I

Materials and Methods

Factor V Peptide Expression and Purification

cDNA for Factor V amino acids 951-1008 was amplified from full-lengthhuman Factor V cDNA using site-specific primers and was subcloned intothe pE-SUMO bacterial expression vector (LifeSensors, Malvern Pa.).SUMO-FV(951-1008) fusion protein was expressed in BL21(DE3) E. coli andpurified with HisTrap™ FF columns (Amersham). After incubating thepurified fusion protein with SUMO Protease (LifeSensors), the FVBRpeptide was purified by ion exchange chromatography. The control peptides46 (derived from the s46 protein construct detailed in Zhu et al.(2007) JBC 282:15033-15039) and the TFPI C-terminal tail peptide(residues 240-276) were generated using a similar approach.

Prothrombin Activation Assays

Steady-state initial velocities of prothrombin cleavage were determineddiscontinuously at 25° C. by measuring the initial rate of thrombinformation. Reaction mixtures contained liposomes (75:25, PC:PS),prothrombin, the indicated Factor V variants, and the Factor V peptidesFVBR or s46. The reactions were initiated by adding Factor Xa, andthrombin generation was determined at multiple time points by monitoringthe cleavage of the chromogenic thrombin substrate S-2238.

Clotting Assays

Residual clotting activity of Factor V variants was determined byprothrombin time-based clotting assays. Factor V variants were incubatedwith either the FVBR or s46 peptide, after which the mixtures were mixedwith Factor V deficient plasma and clotting times were measured. Whereindicated, the Factor V variants were activated by pre-incubation withthrombin prior to addition of the peptides.

Proteolytic Activation of Factor V by Thrombin

Plasma-derived Factor V was incubated with thrombin in the presence orabsence of FVBR peptide. Samples were removed and quenched in samplebuffer at the indicated times, and the cleavage products were analyzedby SDS-PAGE and Coomassie Brilliant Blue staining.

Direct Binding Measurements

Fluorescently-labeled, active site-blocked FXa was incubated with PCPSliposomes and either FV-DT or rFVa in the absence of presence of FVBRpeptide. The ability of the peptide to disrupt binding between FXa andFVDT or rFVa was assessed by monitoring the reduced change in anisotropy(FIG. 3A) or reduction in anisotropy resulting from disruption of theFXa/FVa complex (FIG. 3B).

Results

A Factor V basic peptide (FVBR) having the following sequence wassynthesized: SRAWGESTPLANKPGKQSGHPKFPRVRHKSLQVRQDGGKSRLKKSQFLIKTRKK KKEK(SEQ ID NO: 1), corresponding to amino acids 951-1008 of Factor V. Thepresence of the basic peptide FVBR did not affect activation ofprothrombin by Factor Xa/FVa (FIGS. 2A and 2B). However, a titration ofFVBR does show inhibition of the activation of prothrombin by FXa/FV-DT,where FV-DT is Factor V with amino acids 811-1491 deleted from theB-domain (FIGS. 2A and 2B). FV-DT harbors an important region in thetruncated B-domain which is enriched in acidic amino acids (1493-1537;see FIG. 5). As seen in FIG. 2B, the basic peptide FVBR inhibited FV-DTand similar variants in clotting assays, but not FVa. The other variantstested include s46 (where amino acids 963-1008 from Factor V have beenreplaced with amino acids 1032-1077 from Factor VIII) and FV-1033, whichis a Factor V lacking amino acids 1034-1491. Further, the controlpeptide s46, which corresponds to amino acids 1032-1077 of factor VIII,had no effect. Various FV-810 cleavage mutants were also tested (FIG.2C). Specifically, the arginine at position 709, the arginine atposition 1545, or both were changed to a glutamine to eliminate thethrombin cleavage sites. The ability of FVBR to inhibit these cleavagemutants is shown in FIG. 2C. The data show that the B-domain harboringthe acidic region needs to be tethered to the light chain for the FVBRto effectively inhibit cofactor function. FIG. 2D shows that FVBRprolongs the clot time in normal plasma in a concentration dependentmanner.

The FV basic region peptide was determined to disrupt the binding of FXato FV-DT, but not to FVa. As seen in FIG. 3A, the binding offluorescently labeled FXa to FV-DT on liposomes is impaired in thepresence of FVBR. The disruption of the interaction with FXa by FVBR isobserved only with FV-DT and not FVa (FIG. 3B). FIG. 3C shows the directbinding of FVBR to FVDT, but not FVa.

The FV basic peptide was also determined to delay proteolytic activationof FV by thrombin. In the absence of FVBR, thrombin rapidly cleavesFactor V at Arg709 to produce heavy chain (HC) and then rapidly cleavesat Arg1018 and Arg1545 to produce light chain (LC) (FIG. 4A). In thepresence of FVBR, the cleavage at Arg709 proceeds normally (FIG. 4B).However, the presence of FVBR delayed cleavage at Arg1545 causesaccumulation of B domain+light chain and reduces the amount of lightchain.

In addition to the conserved basic region, the Factor V B domain alsohas a conserved acidic region (FIG. 5). In order to determine if theFactor V acidic domain cooperates with the basic region to stabilizeFactor V in an inactive state, a series of mutants were made.Specifically, B199, B104, and B152 were synthesized. B199 is a FV-DTvariant in which amino acids 963-1008 were inserted between residues 810and 1492; in B104, residues 716-810 from FV-DT were deleted and replacedwith residues 963-1008; in B152, residues 1492-1538 of FV-DT weredeleted and replaced with residues 963-1008. As seen in FIG. 6, thespecific activity of B199 and B104 was dramatically reduced, but theactivity of B152 approached the levels observed for FVa and FV-DT.

The data presented herein demonstrate that the basic region of theFactor V B domain functionally interacts with an acidic region in the Bdomain to stabilize Factor V in an inactive state. The mechanism ofinhibition appears to be mediated at least in part by preventing and/ordisrupting the binding of FXa with Factor V.

A Factor V basic region peptide effectively inhibits Factor V variantsand slows the activation of Factor V by thrombin. These results indicatethat the Factor V basic peptide can be used to regulate thrombingeneration by limiting Factor V activation and cofactor activity.

The C-terminal tail region of TFPI is homologous to FVBR. FIG. 7Aprovides a sequence alignment of FVBR and the C-terminus (amino acids240-265) of tissue factor pathway inhibitor (TFPI). The TFPI peptidedepicted in FIG. 7A was tested for it ability to inhibit FVDT. As seenin FIG. 7B, the TFPI peptide inhibited FVBT in a manner similar to FVBR.FIG. 7C shows that TFPI directly bound FVDT. FIG. 7D shows a competitionassay of unlabeled FVBR or TFPI with Oregon Green® 488 labeled FVBR forFVDT.

EXAMPLE II

Experimental Procedures

Materials

The peptidyl substrateH-D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide (S2238) was fromDiapharma (West Chester, Ohio). Benzamidine,4-amidinophenylmethanesulfonyl fluoride hydrochloride, isopropylβ-D-1-thiogalactopyranoside, BSA, and poly-L-lysine (averageM_(r)=1000-5000) were from Sigma. DansylarginineN-(3-ethyl-1,5-pentanediyl)amide (DAPA) was from HematologicTechnologies (Essex Junction, Vt.). All tissue culture reagents werefrom Invitrogen, except insulin/transferrin/sodium selenite was fromRoche Applied Science. Small unilamellar phospholipid vesicles composedof 75% (w/w) hen egg L-α-phosphatidylcholine and 25% (w/w) porcine brainL-α-phosphatidylserine (PCPS; Avanti Polar Lipids, Alabaster, Ala.) wereprepared and characterized as described (Higgins et al. (1983) J. Biol.Chem., 258:6503-6508).

Proteins

Human prothrombin, thrombin, FX, and FV were isolated from plasma andprepared as described (Baugh et al. (1996) J. Biol. Chem.,271:16126-16134; Buddai et al. (2002) J. Biol. Chem., 277:26689-26698;Katzmann et al. (1981) Proc. Natl. Acad. Sci., 78:162-166; Mann, K. G.(1976) Methods Enzymol., 45:123-156). Recombinant FX (rFX), rFXa, rFVa,plasma-derived FVa, FV-810 (FVDT), and the FV-810 variantsFV-810^(R709Q), FV-810^(R1545Q), and FV-810^(QQ) were prepared,purified, and characterized as described (Camire, R. M. (2002) J. Biol.Chem., 277:37863-37870; Toso et al. (2008) J. Biol. Chem.,283:18627-18635; Toso et al. (2004) J. Biol. Chem., 279:21643-21650).Prethrombin-2 was prepared by proteolysis of human prothrombin andpurified as described (Mann et al. (1981) Methods Enzymol., 80:286-302).Molecular weights and extinction coefficients (E_(0.1%)=280 nm) of thevarious proteins have been reported (Zhu et al. (2007) J. Biol. Chem.,282:15033-15039). All functional assays were performed at 25° C. inassay buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCl₂, and 0.1%polyethylene glycol 8000, pH 7.5).

Construction of FV B-Domain Peptides

BR cDNA-encoding residues Ser⁹⁵¹-Lys¹⁰⁰⁸ of FV was amplified from FVcDNA using primers A (5′-GCAAGGTCTCAAGGTTCACGTGCTTGGGGAGAAAGCACC-3′,forward; SEQ ID NO: 26) and B (5′-GCTTGTCGACTTACTTCTCTTTTTTCTTTTTTCGTGTCTTAATGAGAAACTGG-3′, reverse; SEQ ID NO: 27). The BR+AR peptide(968-1007 and 1492-1538), encoding the juxtaposed BR and AR sequencesfrom the FV variant FVB104, was amplified from FV-B104 cDNA usingprimers C (5′-GCAAGGTCTCAAGG TAGTGGCCACCCAAAGTTTCCTAGAG-3′, forward; SEQID NO: 28) and D (5′-GCTTGTCGACTTAGTTGTCAGGATCTCTGGAGGAGTTGATGTTTGTCC-3′, reverse; SEQ ID NO: 29). Bovine BR (Bos taurusSer⁹³⁸-Lys⁹⁹⁶), green anole BR (Anolis carolinensis Ser¹³³¹-Lys¹³⁹³),and zebrafish BR (Danio rerio Ser¹²⁶⁰-Lys¹³¹⁸) cDNAs were synthesized byGenScript (Piscataway, N.J.). Amplified cDNAs were digested with therestriction enzymes BsaI (5′) and SalI (3′) and ligated into the pE-SUMObacterial expression vector (LifeSensors, Malvern, Pa.), which had beendigested with the same restriction enzymes. All constructs were verifiedby DNA sequencing.

Expression and Purification of B-Domain Peptides

Sequence-verified bacterial expression constructs were transformed intochemically competent BL21 (DE3) cells (EMD Millipore, Billerica, Mass.),and single colonies were used to inoculate liquid LB cultures containing50 μg/ml kanamycin (LB-Kan₅₀). Starter cultures were subcultured into 1liter of LB-Kan₅₀ and incubated at 37° C. until A₆₀₀ reached 0.5, atwhich point, isopropyl β-D-1-thiogalactopyranoside was added at a finalconcentration of 1 mM. After 2 hours, cells were pelleted, resuspendedin lysis buffer (20 mM Tris, 150 mM NaCl, and 1% Triton X-100, pH 8),and lysed with a Misonix Sonicator® 3000 system (Qsonica, Newtown,Conn.). Cell debris was pelleted, and the small ubiquitin-like modifier(SUMO) fusion proteins were purified on HisTrap™ FF columns (GEHealthcare) following the manufacturer's instructions. Purified SUMOfusion proteins were incubated with SUMO protease (LifeSensors) for 2hours at 30° C. to remove the SUMO fusion, and cleaved peptides werepurified by cation exchange chromatography. Protein purity was assessedby SDS-PAGE using 4-12% gradient gels (Invitrogen) in MES buffer,followed by staining with Coomassie Brilliant Blue R-250.

Peptide Acetylation and Fluorescent Labeling

The BR peptide was acetylated by incubation with a 20-fold molar excess(relative to amine groups) of N-hydroxysulfosuccinimide acetate (ThermoScientific) for 1 hour at 25° C. in 20 mM HEPES, 150 mM NaCl, 2 mMCaCl₂, and pH 7.4. Mass spectroscopy data of the acetylated BR peptidewere consistent with quantitative modification of all Lys residues andthe N terminus. Fluorescent labeling of the BR peptide was performed byincubating the BR peptide containing an N-terminal Cys (Cys-BR) for 10min at 25° C. with a 10-fold molar excess oftris(2-carboxyethyl)phosphine HCl (Thermo Scientific) to reducedisulfide bonds, followed by 2 hours at 25° C. with a 20-fold molarexcess of either Oregon Green® 488 maleimide or QSY7 C5-maleimide(Invitrogen). The reactions were quenched by the addition of excess DTT,and labeled BR peptides were purified by gel filtration through Bio-Gel®P-6DG resin (Bio-Rad) to remove excess labeling reagents.

Prothrombin and Prethrombin-2 Activation Assays

Steady-state initial velocities of prothrombin cleavage were determineddiscontinuously at 25° C. as described (Camire, R. M. (2002) J. Biol.Chem., 277:37863-37870). Reaction mixtures containing PCPS (50 μM), DAPA(3 μM), prothrombin (1.4 μM), and either rFVa or the indicated FV-810derivatives were incubated with B-domain peptides (0-10 μM) in assaybuffer. Reactions were initiated with FXa (2 nM), and aliquots werequenched in buffer containing 50 mM EDTA at multiple time points.Prothrombin activation was determined using the chromogenic thrombinsubstrate 52238 as described (Camire, R. M. (2002) J. Biol. Chem.,277:37863-37870). Prethrombin-2 activation was measured similarly toprothrombin using the following reaction conditions: 50 μM PCPS, 1.4 μMprethrombin-2, 3 μM DAPA, 5 nM rFVa or FV-810 derivatives, 1-50 nM FXa,and 0-1000 nM BR peptide.

Clotting Assays

FV derivatives (500 nM) were prepared in assay buffer. Where noted, FVderivatives were pretreated with 10 nM thrombin for 15 minutes at 37°C., followed by the addition of 15 nM hirudin. Samples were diluted to0.25 nM in assay buffer with 0.1% BSA, and the specific clottingactivity was measured in FV-deficient plasma (George King Bio-Medical,Overland Park, Kans.) with TriniCLOT PT Excel (Tcoag, Wicklow, Ireland)as described (Camire et al. (1998) Biochemistry 37:11896-11906). Thedata are presented as the means±S.D.

Fluorescence Anisotropy Measurements

Steady-state fluorescence anisotropy was measured at 25° C. in a PTIQuanta-Master™ fluorescence spectrophotometer (Photon TechnologyInternational, Birmingham, N.J.) using excitation and emissionwavelengths of 480 and 520 nm, respectively, with long-pass filters(KV500, CVI Melles Griot) in the emission beam. Reaction mixtures (2.5ml) containing fixed concentrations (20-40 nM as indicated) of OregonGreen® 488 maleimide-modified BR (OG488-BR) and 50 μM PCPS in assaybuffer were prepared in 1-cm² quartz cuvettes to which increasingconcentrations of FVa or FV-810 were added. Fluorescence anisotropymeasurements, including controls, were performed as described (Buddai etal. (2002) J. Biol. Chem., 277:26689-26698; Betz et al. (1998) J. Biol.Chem., 273:10709-10718).

Analytical Ultracentrifugation

Analytical ultracentrifugation of QSY7 C5-maleimide-modified BR(QSY7-BR) was performed in a Beckman Optima XL-I analyticalultracentrifuge using absorbance optics. Sedimentation velocity wasmeasured at 25,000 or 45,000 rpm with two-sector cells in an An-60 Tirotor at 20° C. Sedimentation of QSY7-BR was followed measuringabsorbance at 560 nm in cells containing 5 μM QSY7-BR alone or with 7 μMFV-810 or rFVa in 20 mM HEPES, 150 mM NaCl, and 2 mM CaCl₂, pH 7.4.Sedimentation coefficients and molecular weights were determined byg(s*) analysis performed using DCDT+ (Philo, J. S. (2000) Anal.Biochem., 279:151-163).

Data Analysis

Data were analyzed by nonlinear least squares regression analysis usingthe Marquardt algorithm (Bevington et al. (1992) Data Reduction andError Analysis for the Physical Sciences, pp. 141-167, McGraw-Hill, NewYork), and the quality of each fit was assessed as described (Straume etal. (1992) Methods Enzymol., 210:87-105). Equilibrium dissociationconstants (K_(d)) and stoichiometries (n) for the interaction betweenOG488-BR and FV-810 in saturation binding measurements were obtainedfrom the change in OG488-BR anisotropy over increasing concentrations ofFV-810, which was corrected for the overall change in fluorescenceintensity (Buddai et al. (2010) J. Biol. Chem. 285:5212-5223;Krishnaswamy, S. (1990) J. Biol. Chem., 265:3708-3718). Displacementbinding experiments, in which unlabeled BR or FXaS195A were titratedinto preformed complexes of OG488-BR FV-810, were analyzed to determineK_(d) and n as described (Betz et al. (1998) J. Biol. Chem.,273:10709-10718). In prothrombin-2 activation reactions, K_(d) valuesfor the interactions of BR and FXa with FV-810 were obtained by globalanalysis of prethrombin-2 activation rates at varying concentrations ofFXa and BR fit to a model of tight binding using DYNAFIT (Kuzmic, P.(1996) Anal. Biochem., 237:260-273).

Results

Inhibition of Cofactor-Like FV Variants by B-Domain Fragments

As explained hereinabove, a minimal inhibitory motif has been identifiedwithin the FV B-domain that consists of evolutionarily conserved basicand acidic elements. To define how these elements inhibit FV function,B-domain fragments were expressed as SUMO fusions in Escherichia coliand purified by ion exchange chromatography following removal of theSUMO tag. As explained hereinabove, the inhibitory effects of thepurified fragments were determined in assays containing either FVa orthe cofactor-like FV variant FV-810. In reconstituted prothrombinactivation reactions, the BR peptide effectively inhibitedprothrombinase containing FV-810 (see above). The clotting activity ofFV-deficient plasma supplemented with FV-810 was similarly inhibited inthe presence of the BR peptide (FIGS. 2D and 8), indicating that the BRpeptide reconstituted a functional inhibitory procofactor regulatoryregion (PRR) within FV-810. In contrast to FV-810, the BR peptide had noinhibitory effect on rFVa in prothrombin activation assays or clottingassays (FIGS. 2 and 8). Furthermore, whereas a minimal B-domain almostexclusively composed of tandem BR and AR elements is sufficient tostabilize FV as a procofactor, rFVa was not inhibited by the BR+ARpeptide (FIG. 8B). Thus, whereas the BR can act in trans to reconstitutethe PRR, it appears that the acidic region (AR) must be covalentlyattached to mediate inhibition of procoagulant activity.

The complementary charge states of the BR and AR elements suggest theelectrostatic forces likely contribute to PRR function. Indeed, the BRpeptide that had been acetylated to neutralize the positive charge nolonger inhibited FV-810 (FIG. 8A). However, positive charge alone wasinsufficient to reconstitute a functional PRR, as neither low molecularweight poly-L-lysine (average M_(r)=1000-5000) (FIG. 8A) nor thecationic platelet factor 4 affected FV-810 activity. Thus, inhibition ofFV by the PRR is both charge- and sequence-dependent.

Direct Binding of the BR Peptide to FV Variants

The ability of the BR peptide to inhibit FV-810 suggests that thepeptide binds to FV-810 to reconstitute a functional PRR. To test this,direct binding of the BR peptide to FV-810 or rFV was measured usingmultiple approaches. First, the BR peptide was fluorescently labeledwith Oregon Green® 488 maleimide, and changes in fluorescence anisotropywere monitored. Titration of FV-810 into reactions containing fixedconcentrations of OG488-BR produced saturable binding curves (FIG. 9)with calculated equilibrium binding values of K_(d)=2.07±0.2 nM andn=1.27±0.02 mol of FV-810/mol of OG488-BR. The binding of OG488-BR toFV-810 was calcium-dependent, as no binding was observed when 10 mM EDTAwas added to the buffer. Titrating the unlabeled BR peptide intoreactions containing a preformed complex of OG488-BR and FV-810 reducedthe anisotropy signal toward the base line (FIG. 9, inset). From thesedisplacement curves, the equilibrium binding values for the unlabeled BRpeptide were calculated to be K_(d)=2.1±0.2 nM and n=1.0±0.06 mol ofBR/mol of FV-810, essentially identical to the labeled peptide. Incontrast to FV-810, rFVa showed no detectable binding to OG488-BR (FIG.9).

As a complementary approach, binding between the BR peptide and FV-810was also monitored by analytical ultracentrifugation. Sedimentationvelocity experiments were performed with 5 μM QSY7-BR either alone (FIG.10A) or with 7 μM FV-810 (FIG. 10B). In the absence of FV-810, thesedimentation coefficient (s⁰ _(20,w)) of QSY7-BR was 0.98 s, identicalto the value for recombinant hirudin, which is similar in size (Otto etal. (1991) Eur. J. Biochem., 202:67-73). In the presence of FV-810, theQSY7-BR sedimentation coefficient shifted to 9.8 s, somewhat larger thanthe determined value for FV-810 of 8.43 s (Toso et al. (2004) J. Biol.Chem., 279:21643-21650). This increase in the sedimentation coefficientis consistent with a 1:1 stoichiometry between QSY7-BR and FV-810,agreeing with the calculated stoichiometry from fluorescencemeasurements. Molecular weight determination yielded Mr=7210±40 for theQSY7-labeled peptide and Mr=199,000±3000 for QSY7-BR-FV-810 complex,which are in agreement with expected values. QSY7-BR sedimentationvelocity was also performed with rFVa or with FV-810 in buffercontaining 10 mM EDTA as controls. The data from these controlexperiments indicated a weak interaction (K_(d)≥1 μM) consistent withthe anisotropy data showing that no detectable binding was observedusing ≤100 nM FVa (FIG. 9).

PRR Sequence Specificity

To assess the sequence specificity of the PRR, BR fragments weregenerated from several vertebrate species and their effect on FV-810 wascompared with that of the human BR. The bovine BR, which is highlyconserved with the human BR (FIG. 1), inhibited FV-810 in bothprothrombin activation reactions and clotting assays compared with thehuman BR, whereas the more divergent BR fragments from lizard (A.carolinensis) and zebrafish (D. rerio) had no effect on FV-810 activity(FIG. 11A). In displacement binding experiments, the bovine BR exhibited˜10-fold weaker binding to FV-810 than did the human BR (K_(d)=28.3±0.6versus 2.2±0.2 nM, respectively) (FIG. 11B).

Competition Between the BR and FXa

A fundamental difference between procofactor-like and cofactor-like FVproteins is the ability of the latter to bind to FXa with high affinity.As demonstrated herein, a minimal B-domain consisting almost exclusivelyof the PRR is sufficient to maintain the procofactor state. The PRRlikely occludes a high affinity FXa-binding site on FV; thus, inhibitionof FV-810 procoagulant function by the BR peptide could reflectcompetitive binding between the BR and FXa to FV(a). Consistent withthis, when catalytically inactive FXaS195A was titrated intofluorescence binding assays, it displaced OG488-BR from FV-810 (FIG.12A). Using the previously determined binding constants for OG488-BR, itwas calculated that FXa^(S195A) bound to FV-810 with K_(d)=1.8 nM andn=1.1 mol FXa/mol of FV-810, consistent with the reported equilibriumbinding values for FXa and FV-810 (Toso et al. (2004) J. Biol. Chem.,279:21643-21650). As a control, titration of zymogen FXS195A atconcentrations up to 1 μM had little effect on OG488-BR anisotropy (FIG.12A).

The competition between the BR peptide and FXa was also assessed bymonitoring prethrombin-2 activation in reactions containing variableconcentrations of FXa and the BR peptide at a fixed concentration ofFV-810. Under these reaction conditions, the BR and FXa followed a modelof competitive binding (FIG. 12B), with fitted equilibrium bindingvalues K_(d)=2.0±0.2 nM for FXa and K_(d)=34.2±3.6 nM for the BR. Thecalculated K_(d) for the BR from these reactions is somewhat higher thanthat observed in direct binding measurements (FIG. 9). This discrepancymay be due to the generation of FVa in the reactions from feedbackproteolysis of FV-810 by thrombin. Together with the fluorescencebinding data, these results indicate a model of competitive binding ofthe BR and FXa to FV-810.

Role of Site-Specific Proteolysis in PRR Stability and Inhibition

The observation that rFVa activity is not inhibited by any of theB-domain peptides suggests that proteolysis of the FV B-domain bythrombin irreversibly disrupts the PRR, thereby producing the activecofactor FVa. The effect of proteolysis was assessed at individualthrombin cleavage sites on PRR function by comparing the ability of theBR peptide to inhibit FV-810 cleaved by thrombin at either Arg709 orArg1545. FV-810 variants containing the R709Q or R1545Q mutation werepreincubated with thrombin to generate cleavage products in which theB-domain was still tethered to either the heavy and/or light chains(FIGS. 2C and 13). Prior to thrombin cleavage, all FV-810 variants werepotently inhibited by the basic peptide (FIGS. 2C and 13). However,following incubation with thrombin, only FV-810^(R1545Q) and FV-810^(QQ)were still inhibited by the BR peptide (FIGS. 2C and 13). Furthermore,when binding of the thrombin-cleaved FV-810 variants to OG488-BR wasmeasured by fluorescence anisotropy, only FV-810^(R1545Q) andFV-810^(QQ) still bound OG488-BR, whereas FV-810 and FV-810^(R709Q) didnot (FIG. 14). Thus, cleavage of FV at Arg1545 specifically disrupts theability of the BR to bind to FV-810 and establish a functional PRR.Interestingly, cleavage at Arg709 appears to partially destabilize thePRR, as FV-810^(R1545Q) displayed a weakened binding affinity forOG488-BR after thrombin cleavage (K_(d)=30 versus 2 nM). Thisobservation indicates that proteolytic activation of FV progressivelydisrupts the structural integrity of the PRR, thereby reducing itsinhibitory effect.

In summary, the data presented herein provide mechanistic insight toexplain how the B-domain stabilizes FV as an inactive procofactor andhow discrete proteolysis of FV generates an active cofactor for FXa. Thebipartite PRR, composed of cis- and trans-acting acidic and basicelements, respectively, suppresses cofactor activity by competitivelyinhibiting FXa binding to FV. Discrete proteolysis of the B-domain bythrombin destabilizes the PRR, with cleavage of the Arg¹⁵⁴⁵ peptide bondcausing irreversible dissolution of PRR integrity and fully exposing ahigh affinity FXa-binding site. The ability of the BR to function as atrans-acting inhibitor of FV(a) activity offers a novel target formodulating FV(a) activity in vivo. Specifically, ligands that mimic theBR will function as anticoagulants by opposing or delaying full FVactivation in the early stages of coagulation.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

What is claimed is:
 1. An isolated peptide selected from the groupconsisting of: i) a peptide having at least 80% homology with SEQ ID NO:1, 2, or 3, wherein said peptide has a length of about 20 to about 80amino acids and ii) a peptide consisting of SEQ ID NO:
 4. 2. Theisolated peptide of claim 1, wherein said peptide has at least 90%homology with SEQ ID NO: 1, 2, or
 3. 3. The isolated peptide of claim 1,wherein said peptide comprises SEQ ID NO: 1, 2, or
 3. 4. The isolatedpeptide of claim 1, wherein said peptide comprises SEQ ID NO:
 3. 5. Theisolated peptide of claim 1, wherein said peptide comprises at least onemodification selected from the group consisting of amidation andacetylation.
 6. A composition comprising at least one peptide of claim 1and at least one pharmaceutically acceptable carrier.
 7. The compositionof claim 6, further comprising at least one anti-thrombosis compound. 8.The isolated peptide of claim 1, wherein said peptide has a length ofabout 20 to about 60 amino acids.
 9. The isolated peptide of claim 1,wherein said peptide comprises at least one D-amino acid in place of anL-amino acid.
 10. The isolated peptide of claim 1, wherein said peptideconsists of SEQ ID NO: 1, 2, 3, or
 4. 11. The isolated peptide of claim1, wherein said peptide consists of SEQ ID NO: 4.